Canine monocytic ehrlichiosis (CME) is a tick-borne rickettsial disease, affecting dogs and other canine species worldwide. The causative agent is Ehrlichia canis, which infects circulating lymphocytes and is transmitted transtadially by the brown dog tick Rhipicephalus sanguineus. CME occurs in acute and subclinical phases, and in some cases progresses to a chronic phase. The acute phase is characterized by fever, anorexia, depression, lymphadenopathy and mild thrombocytopenia. Dogs typically recover from the acute illness, but become persistently infected carriers of the organism without clinical signs of disease for months or even years. When it develops, the chronic phase is characterized by clinical signs including thrombocytopenia, hyperglobulinemia, anorexia, emaciation, and hemorrhage, particularly epistaxis, petechiae and ecchymoses, and may result in death. Pancytopenia characterizes the chronic phase of the disease and may persist for a period of months, or for the remaining life of the dog. Supportive therapy is often necessary to combat wasting and specific organ dysfunction.
Human monocytic ehrlichiosis (HME) is caused by the related organism E. chaffeensis, and is characterized by fever, headache, myalgia and leukopenia. Furthermore, a similar disease termed Venezuela Human Ehrlichiosis (VHE) has been determined to be caused by a strain of E. canis designated VHE AF373612 (Perez et al. (2006) Ann N Y Acad Sci. 1078:110-7). Antibiotic therapy, particularly with doxycycline or tetracycline, is used for treatment of CME, HME and VHE, and is mainly effective if initiated at an early disease stage. There is no commercial vaccine for any of the aforementioned diseases, and tick control remains the recommended prophylactic method.
A number of proposed vaccines against E. canis and methods of protection against E. canis infection are known in the prior art.
US Patent application publication Nos. 2005/0202046 and 2006/0188524 disclose a vaccine composition which comprises an inactivated E. canis bacterin, a carrier, and an adjuvant system; and a method for the prevention or amelioration of canine ehrlichiosis in a dog by administration of such a vaccine. The disclosed adjuvant system contains both an antibody response inducing agent e.g. EMA/NEOCRYL® (Ethylene Maleic Anhydride (EMA)/Neocryl), and a cell-mediated immunity response inducing agent e.g. Bacilli Calmette-Guerin (BCG) or EMULSIGEN (Oil-in-Water Emulsified Adjuvant). While these publications allege that the invention effectively induces immunity, the disclosed vaccines are in fact deficient, since a significant proportion of dogs immunized with the vaccines are reported to develop canine ehrlichiosis. In addition, preparation of such vaccines is costly, due to the inclusion of multiple adjuvants, and the requirement for inactivating the E. canis strains. Inactivation, according to these disclosures, is carried out by conventional means, including use of chemical inactivating agents such as binary ethyleneimine, beta-propiolactone, formalin, merthiolate, gluteraldehyde, sodium dodecyl sulfate, or mixtures thereof; heat, or psoralen in the presence of ultraviolet light.
Other prior art disclosures relate to vaccines comprising isolated E. canis antigens or combinations thereof, for example as disclosed in US Patent Application Publication No. 2006/0234322. U.S. Pat. Nos. 6,458,942 and 6,392,023 disclose recombinant forms of homologous 28-kDa immunoreactive proteins of E. canis, and a method of inhibiting E. canis infection by administration of a composition comprising such antigens. US Patent Application Publication No. 2004/0170972 discloses the E. canis recombinant proteins ProA, ProB, mmpA, and a cytochrome oxidase homolog, and use thereof for a recombinant vaccine. US Patent Application Publication No. 2004/0126871 discloses a DNA vaccine comprising the gene for major antigenic protein-1 (MAP-1) and/or MAP-2 of Rickettsia rickettsii, and a method for protecting a host against disease or death caused by a rickettsial pathogen, including E. canis, by administration of such a vaccine. US Patent Application Publication No. 2004/0121433 discloses a canine ehrlichiosis vaccine, which comprises the E. canis immunoreactive surface protein p153, or the E. chaffeensis immunoreactive surface protein p156. U.S. Pat. No. 6,306,394 discloses a vaccine comprising a granulocytic ehrlichia protein or a fragment thereof, and a method of preventing ehrlichiosis in an animal by administration of such a vaccine. The aforementioned vaccines and methods are not directed to the full complement of cell surface antigens of E. canis, and accordingly they may not be completely effective in preventing ehrlichiosis in challenged hosts, since they offer the possibility of evasion of immune response by the infectious agent, even in vaccinated hosts.
Accordingly, there remains an unmet need in the art for an effective vaccine against E. canis. 
E. canis strain Israel 611 has been propagated in vitro in a continuous canine macrophage cell line, DH82 (Keysary et al. (1996) Vet Parasitol 62:331-40). The same strain has alternately been propagated in a continuous mouse macrophage cell line, J774.A1 (Keysary et al. (2001) J Vet Diagn Invest 13(6):521-3). U.S. Pat. No. 5,192,679 discloses a method for continually growing a bacterial pathogen comprising infecting DH82 with E. canis and cultivating the infected cells in a suitable culture medium. The prior art does not teach or suggest an attenuated strain of E. canis suitable for use as a vaccine against disease caused by pathogenic strains.